Various systems are available for expressing proteins in bacteria, and heterologous proteins are typically produced at high levels using E. coli as a host. However, E. coli is not always the most desirable host bacterium--in certain circumstances it is particularly advantageous to use Bacillus subtilis. The advantages of B. subtilis as an expression host include its non-pathogenicity, absence of significant codon bias (Brown, T. A., 1991, Genomes and Genes, In: T. A. Brown (Ed.), Molecular Biology Labfax, BIOS Scientific Publisher Ltd., Oxford, pp. 235-254), the presence of secretory mechanisms, its extensively studied genetics, and the facility of large-scale manipulation using standard protocols (Simonen, M. and Palva, I., 1993, Microbiological Reviews, 57: 109-137).
To date, protein expression using B. subtilis has been unsuccessful due to low product expression levels. This has resulted from the lack of efficient regulatory elements, both for transcriptional and post-transcriptional control of expression. One major problem encountered in B. subtilis is that at the onset of the stationary (S) growth phase it expresses large quantities of proteases which are detrimental to the integrity of the heterologous protein which is being expressed and therefore product expression during S-phase is unacceptably low. Efforts have been made to address this (Wong, S. L., 1995, Current Opinion in Biotechnology, 6: 517-522). This problem has been overcome in the past by the use of protease-deficient and endonuclease-defective strains. However, although able to express desired proteins at improved levels during S-phase, they suffer from the problem of typically being slow-growing strains and introducing additional expense and difficulties into the manufacturing process.
It has previously been suggested (Lam, K. H. E., Chow, K. C. and Wong, W. K., Abstract T3.36, Asia Pacific Society of Bioscientists, Second International Symposium and Workshop, Jul. 8-11 1996) to express proteins in B. subtilis during the vegetative growth phase (VGP) using an expression/secretion cassette comprising the B. subtilis veg promoter, the E. coli lac operator, the Staphylococcal protein A leader sequence for secretion, a multiple cloning region, translational stop codons and the efficient B. subtilis gnt transcriptional terminator. The cassette may be cloned into a modified B. subtilis/E. coli shuttle vector pRB373M2 to form an expression/secretion vector named the veg vector. The endoglucanase (Eng) gene and the human epidermal growth hormone gene (hEGF) were stated as having been cloned into the veg vector.
The present inventors have now succeeded in making a novel expression cassette, particularly for use in B. subtilis and E. coli, which allows very high expression levels of heterologous proteins to be achieved in B. subtilis.
Previously, the use of the vegI promoter alone in B. subtilis has not been described--the use of the veg(comprising the vegI and vegII promoters) (see for example U.S. Pat. No. 4,783,405; U.S. Pat. No. 4,710,464; U.S. Pat. No. 4,559,300) and vegII (see for example Le Grice, F. J., 1990, Methods Enzymol., 185: 201-214) promoters in heterologous protein expression has been described. As a result of the isolation and purification of an unexpected vegI mutant occurring at very low levels (a total of five B. subtilis transformants were found to harbour plasmid DNA containing the mutant vegI promoter in a transformation in which about 300 .mu.g of DNA was used), it has now been found that a B. subtilis endonuclease activity appears to act upon the vegI promoter, causing digestion and subsequent exonuclease digestion of the promoter. The present inventors have succeeded in isolating the putative vegI promoter B. subtilis endonuclease restriction site, allowing the modification of the palindromic restriction site to protect against B. subtilis endonuclease activity and allow the use of the modified vegI promoter. One particular modified vegI promoter is described below but other modifications may be readily made to the vegI promoter to protect against the endonuclease activity whilst still retaining the promoter functionality. This allows the use of the vegI promoter alone with B. subtilis, not previously suggested by the prior art. The identification of the palindromic endonuclease restriction site is particularly surprising since it is an octameric sequence, whereas most endonuclease restriction sites are hexamers.
According to the present invention there is provided an endonuclease-protected vegI promoter, and particularly a B. subtilis endonuclease-protected vegI promoter.
The present inventors have found that the octameric sequence of SEQ ID NO: 20, which forms part of the -10 region of the vegI promoter, is a B. subtilis endonuclease restriction site, meaning that constructs containing the vegI promoter undergo endonuclease (and subsequently, exonuclease) digestion when transformed into B. subtilis. This in turn results in the construct failing to express any coding sequences it contains.
The identification of the endonuclease restriction site has allowed the development of endonuclease-protected vegI promoters which are protected against endonuclease restriction digestion, yet which still retain their promoter functionality. Specifically, the fifth residue of SEQ ID NO: 20 may be substituted from A to G to give the endonuclease-protected sequence of SEQ ID NO: 21. The endonuclease restriction site is typically found at nucleotides -15 to -8 of the vegI promoter relative to the transcription start nucleotide beginning at nucleotide +1 (nucleotide 51 of SEQ ID NOs: 12 and 13) (Le Grice, S. F. J. et al., 1986, Mol. Gen. Genet., 204: 229-236). An example of an endonuclease-protected vegI promoter is that of SEQ ID NO: 12. The sequence of a typical vegI promoter is that of SEQ ID NO: 13.
Alternatively endonuclease-protection may be achieved by other nucleotide substitutions to the octamer. The substitutions may be simply made and the efficacy of the substituted readily determined using the experimental procedures detailed below. Alternatively, nucleotides of the restriction site may be methylated using primers with specifically methylated nucleotides and the standard procedures of PCR and subcloning. Other substitutions will be readily apparent to one skilled in the art and may be readily made and the efficacy of modified promoters simply determined.
Also provided according to the present invention is a DNA construct for expressing a coding sequence in B. subtilis, comprising operatively linked in the 5' to 3' direction:
a) an endonuclease-protected vegI promoter; PA1 b) a DNA coding sequence encoding an RNA encoding an expression product; and PA1 c) a 3' non-translated region. PA1 a) an endonuclease-protected vegI promoter; PA1 b) at least one cloning site into which may be inserted a DNA coding sequence encoding an RNA encoding an expression product; and PA1 c) a 3' non-translated region. PA1 a) transforming a bacterium with a DNA construct according to the present invention containing the coding sequence of said gene for said expression product; PA1 b) culturing said bacterium to cause expression of said coding sequence; and PA1 c) isolating and purifying said expression product. PA1 a) the vegI promoter; PA1 b) a DNA coding sequence encoding an RNA encoding an expression product; and PA1 c) a 3' non-translated region. PA1 a) culturing a transformed E. coli according to the present invention, said E. coli having been transformed with a DNA construct containing the coding sequence of said gene for said expression product, said culturing causing expression of said coding sequence; and PA1 b) isolating and purifying said expression product.
It may additionally comprise between said endonuclease-protected vegI promoter and said DNA coding sequence a lac operator, ribosome binding site, and SPA leader sequence. Said 3' non-translated region may comprise a stop codon and the gnt transcriptional terminator.
Said DNA coding sequence may comprise the coding sequence for a heterologous protein, for example the Cellulomonas fimi cenA coding sequence or the human epidermal growth factor (hEGF) coding sequence. The successful results obtained (below) expressing both hEGF and endoglucanase (Eng), two very different heterologous proteins, shows that such a construct is capable of successfully expressing a wide range of proteins. Other heterologous proteins which may be expressed using the construct of the present invention include human interleukin 1 (Bellini, A. V. et al., 1991, J. Biotech. 18: 177-192) and the antidigoxin single-chain antibody (Wu, X. C. et al., 1993, Bio/Technology, 11: 71-76). Other proteins will be readily apparent to one skilled in the art.
Also provided according to the present invention is a DNA construct for expressing a coding sequence in B. subtilis, comprising operatively linked in the 5' to 3' direction:
In such a construct, the cloning site may comprise a multiple cloning site. The construct may additionally comprise between said endonuclease-protected vegI promoter and said cloning site a lac operator, ribosome binding site, and SPA leader sequence. Said 3' non-translated region may comprise a stop codon and the gnt transcriptional terminator. An example of such a construct is that detailed in FIG. 1.
Also provided according to the present invention is a bacterium transformed with a DNA construct according to the present invention. Experiments (below) detail B. subtilis transformed with such a construct. Experiments have also shown that expression can be successfully achieved in E. coli. Successful expression my also be achieved in other bacteria, for example Staphylococcus aureus, the pRB373 shuttle vector (see below) having been derived from the S. aureus plasmid pUB110 (Gryczan, T. J. et al., 1978, J. bacterial., 134: 318-329).
Also provided according to the present invention is the expression product of a DNA construct according to the present invention made by a bacterium transformed with same. Such an expression product may be isolated and purified.
Also provided according to the present invention is a method of manufacture of an expression product of a gene, comprising the steps of:
As detailed above, experiments have shown that E. coli transformed with a construct having an endonuclease-protected vegI promoter are capable of successfully expressing the coding sequence of the construct. It has now also been found that, surprisingly, E. coli transformed with a construct having an unmodified vegI promoter are also capable of expressing the coding sequence of the construct. Thus the present invention also provides an E. coli bacterium transformed with a DNA construct for expressing a coding sequence, said DNA construct comprising operatively linked in the 5' to 3' direction:
Such a use of a vegI promoter has neither been suggested nor disclosed by the prior art.
Also provided according to the present invention is the expression product of a DNA construct made by an E. coli bacterium according to the present invention, transformed with said DNA construct.
Also provided according to the present invention is a method of manufacture of an expression product of a gene, comprising the steps of:
Also provided according to the present invention is a vegI promoter having a guanine nucleotide substituted at position -11 relative to the transcriptional start nucleotide beginning at nucleotide +1.
The invention will be further apparent from the following description, with reference to the several figures of the accompanying drawings, which show, by way of example only, form of B. subtillis expression systems.